The 3'untranslated regions (UTRs) of messenger RNAs contain cis-regulatory elements that affect gene expression by altering mRNA stability and translation. Two large classes of 3'UTR cis-regulatory elements have been described: AU-rich elements and miRNA target sequences. Current predictions suggest that many 3'UTRs contain AU-rich elements or miRNA targets but only a very small fraction of these predictions have been experimentally verified. There are also other classes of 3'UTR cis-regulatory sequences, including RNA localization elements ('zip codes') and natural antisense targets. Systematic genome-wide approaches are likely to uncover many more examples of different classes of 3'UTR cisregulatory elements. High-throughput technologies for systematically identifying 3'UTRs with cisregulatory activity and mapping the functional elements within these 3'UTRs could dramatically accelerate progress in this field. We are developing a novel reporter system that can be used to identify effects of 3'UTR sequences on mRNA stability and translation. Since the system relies on lentiviral transduction of the reporter constructs, it can easily be applied to a range of cell types where different trans-regulatory factors are active. The system is designed for use with complex libraries of full-length 3'UTR sequences or shorter 3'UTR sequence elements. By using flow cytometry and microarrays, it will be possible to simultaneously assess the effects of large numbers of 3'UTR sequences on mRNA stability or translation in a single assay. This proposal has two specific aims. Aim 1 will validate the ability of the novel reporter system to measure effects of individual 3'UTRs on mRNA stability and translation. Aim 2 will establish and validate high-throughput multiplex assays for analyzing 3'UTR activity and mapping functional elements within 3'UTRs. The tools developed here could be used to discover and analyze 3'UTR regulatory elements throughout the genome and will be made available to all interested investigators. Used together with other existing experimental and bioinformatics approaches, these new tools will help produce novel and comprehensive information about how 3'UTRs high regulate gene expression in health and disease.